Although a central role of human papillomavirus (HPV) in risk for cervical cancer and its precursor, cervical intraepithelial neoplasia grades ll-lll or carcinoma in situ (greater than or equal to CIN2-3) has been well established, most of the HPV infections resolve spontaneously and only few progress to cervical cancer. Thus, an HPV-based screening usually suffers from a low specificity for identification of women with SCIN2-3. Clearly, factors other than HPV infection also play an important role in pathogenesis of cervical neoplasia. A loss of function of certain critical genes by methylation of CpG islands in promoter region has been found in a variety of cancers, including cancer of the cervix. Almost every tumor type appears to have CpG hypermethylation in multiple genes and a unique hypermethylation profile exists for different tumors. We hypothesize that aberrant DNA methylation is associated with risk of cervical neoplasia and its detection in the exfoliated cell sample obtained at the routine screening can serve as a biomarker for identification of women who are at high risk of greater than or equal to CIN2-3. We propose to efficiently address our hypotheses by performing additional analyses on a subset of samples collected in the ongoing NCI-funded project entitled "Evaluation of Cervical Cancer Screening Methods (EVA)," a study designed to evaluate a variety of screening strategies for detection of greater than or equal to CIN3. Interview data, cytology and histology, and HPV results will be available to the planned study. We plan to define a panel of genes that are closely related to risk of greater than or equal to CIN2-3 (Aim one) by mapping methylation patterns of CpG islands of the 12 or more candidate genes between women with greater than or equal to CIN2-3 (cases) and those with CIN1 or normal histology (controls). Based on the methylation patterns between the cases and controls, we will design a high throughput assay (MethyLight) for detecting the hypermethylated genes in cervical swab samples. In Aim two, we will examine performance of testing the panel of hypermethylated genes for identification of women with greater than or equal to CIN2-3. Lastly (Aim three), among women with HPV16 infection, we will determine whether CpG hypomethylation in the long control region of HPV16 genome is associated with an increased risk of greater than or equal to CIN2-3, high level of E7 mRNA, and repeated HPV16-positive visits. Data from the proposed study will not only shed light on our understanding of the role of CpG methylation in development of cervical neoplasia but also help to better design future cervical cancer control programs.